Device And Method For Separating, Mixing And Concentrating Magnetic Particles With A Fluid And Use Thereof In Purification Methods

ABSTRACT

The present invention is related to a process for manipulating magnetic particles that are suspended in a fluid, possibly containing a biological entity of interest, the magnetic particles being able to bind the entity of interest, the fluid being contained in a reaction vessel constituted by a large upper compartment with a funnel shape, an elongate lower compartment with a substantially constant cross-section and a closed base. The process consists of: a) subjecting the magnetic particles to two magnetic fields applied simultaneously to separate all said magnetic particles present in at least the upper compartment of the vessel from the fluids b) transferring the separated magnetic particles from the upper compartment to the elongated lower compartment, c) removing the fluid from the vessel, d) adding a washing liquid to the lower compartment), e) subjecting the rest of the fluid to at least two magnetic fields applied successively with different and changing directions to wash all the magnetic particles present in the lower compartment, and concentrating said magnetic particles in said lower compartment The according to the present invention are especially useful in methods for the extraction of nucleic acid to enable them for further processing.

This invention relates to the use of magnetic or magnetizable particles,and, in particular, to methods of separating, mixing and concentratingmagnetic or (super) paramagnetic particles efficiently with a fluid andoptionally followed by resuspension of the particles in another fluid.The invention further provided a device for doing the same. In thebelow-exposed invention, the magnetic particles, paramagnetic particlesand superparamagnetic particles will be called magnetic particles.

State of the Art Concerning the Separation Step:

In many methods of biological analysis, a solid phase has to beseparated from a liquid phase and subsequently washed. To wash the solidphase, a defined amount of buffer solution is pipetted into the reactionvessel containing the solid phase to suspend the solid phase in thebuffer solution. The solid and the liquid phases are then separated. Theliquid phase is then removed by suction (aspiration) and a new washingprocess begins. Usually a number of washing cycles are carried out, eachincluding a suspension, separation and aspiration process.

The use of magnetic particles as a solid phase and separation bypermanent magnets is known in principle. Permanent magnets attract theparticles to the wall of the reaction vessel and hold them there.

Magnetic particles are often used in separation processes. There aremany biological assay methods and purification methods in which magneticparticles are used. For example, immunoassay methods, nucleic acidhybridisation assays and the like. Magnetic particles can also be usedin purification methods, to isolate particular components, proteins,nucleic acids, from the material in which they were contained. Theparticles can be used to separate certain components from a mixture, forexample, because they are coated with a reagent with a specific affinityfor the component. Magnetic particles can be drawn to, for example, thewall of a container in which the fluid with the magnetic particles wascontained and the fluid can be removed and, optionally, be replaced withanother fluid. Thus, the particles can be mixed with the fluid fromwhich the specific component is to be removed, the component will bindto the magnetic particle, and a magnet can be used to separate theparticles with the component from the remainder of the mixture in thefluid. Optionally the magnetic particles can be washed, and can beseparated in another fluid. Or the component can be removed from theparticles again into another fluid.

European patent application EP-A-0.136.126 describes a device forseparation during solid-phase immunoassays. The bottom end of a reactionvessel containing magnetic particles is disposed between two permanentmagnets. The axes of magnetization are at right angles to the wall ofthe reaction vessel, thus reducing stray magnetic fields.

International application WO-A-92/05443 describes a device forseparating magnetic particles. The reaction vessels containing themagnetic particles are disposed in rows. Between the rows is positioneda magnetic block. The reaction vessels are disposed in the magneticblock such that two magnets-are diametrically opposite relative to thereaction vessel. The magnets have alternating polarities and theirmagnetization axes extend parallel. Separated particles are on only oneside of the reaction vessel.

The U.S. Pat. No. 4,895,650, the contents of which is hereinincorporated by reference, describes a separating device in whichparticles are separated by a permanent magnet. The magnet is on only oneside of the reaction vessel. The relation between the level of testsolution in the test-tube and the position of the magnet is focused on.The position of the magnet, more particularly its height, must coincidewith the level of test solution in the reaction vessel, and is broughtto the desired height by packing material in the bottom part of thedevice holding the magnet.

During an immunoassay, the fluid level in the reaction vessels afteradding the required reagents is not necessarily uniform. For example,the level in the reaction vessel after adding conjugate solution may belower than the level after adding washing buffer solution. The method ofanalysis described in this former U.S. Pat. No. 4,895,650 does not takethese differences in level into account.

Known devices for separating magnetic particles have the disadvantage ofrequiring a relatively long time before all magnetic particles areseparated from the liquid phase. Separation time may be considerable,particularly for larger volumes.

A device for rapid separation of magnetic particles is described inEuropean patent application EP-A-0.317.286. In this device, the reactionvessel is surrounded by four permanent magnets (magnets 1, 2, 3 and 4),which are uniformly distributed around a reaction vessel. The directionof the magnetic field of magnets 1 and 3 is rotated through 180°relative to the direction of the magnetic field of magnets 2 and 4. Thisdevice has the disadvantage of requiring a relatively large number ofpermanent magnets to speed up separation. It also excludes many possiblecell movements.

In the EP-B-0.644.425 patent is presented an analyser having a devicefor separating magnetic particles from a suspension, the separationdevice containing two permanent magnets between which the reactionvessel containing a suspension is located. For faster and more completeseparation of the magnetic microparticles, the magnets are locateddiametrically opposite with respect to the reaction vessel and the poleaxes of the magnets form an acute angle with the longitudinal axis ofthe reaction vessel. An aim of this invention is to provide ananalytical device comprising a device for separating magnetic particlessuch that the magnetic particles in suspension can be rapidly separatedeven when the reaction vessel is filled to different levels. Another aimis to provide an analytical device for separating magnetic particlessuch that the magnetic particles in suspension can be separated in afocused manner.

If these documents can be considered for the separation of magneticparticles from a liquid of interest, they cannot authorize a performantmixing that could efficiently wash the particles and give them allchance to bind onto the magnetic particle's surfaces. This efficientstep of mixing is absolutely necessary for purifying nucleic acidtargets from a biological sample.

State of the Art Concerning the Mixing Step:

Purification methods for nucleic acid using magnetic particles have forexample been described in various applications such as EP-A-0.757.106and WO-A-96/41811. In these applications methods are described wherein asample solution containing nucleic acids is treated with a chaotropicsubstance to release the nucleic acid. After releasing the nucleic acidsfrom the biological entity in the lysis buffer, the nucleic acids arebound to the magnetic particles. Both particles coated with atarget-specific probe as well as particles having a metal oxide coating(e.g. silica), giving a generic binding of all nucleic acids containedin the sample are used for this purpose. After binding the target,interfering components such as cell debris, enzymes, proteinsanti-coagulants and salt are removed by washing the magnetic particlesin a (set of) wash buffer(s). Finally, the purified nucleic acids arereleased from the particles by mixing the particles in a small volume ofelution buffer.

For efficient washing and elution the magnetic particles need to be welldispersed and mixed in the relevant buffers. In general, this washingand elution process may be hampered by the aggregation or clogging ofthe magnetic particles either caused by the adsorption of specificcomponents in the lysed sample (e.g. genomic DNA) or by residualmagnetic dipole fields induced in the particles. In particular, the useof silica coated (magnetic) particles with samples that containsignificant amounts of genomic DNA (whole blood, sputum, tissue),results in a tight pellet that is difficult to process.

Well-known methods for mixing (magnetic) beads in a liquid buffer arevortexing, sonification or pipetting. These methods however aredifficult to automate, and/or give risk of sample to samplecontamination by aerosol generation or they may degrade the nucleic acidtarget. Furthermore, these methods are not well suited for very smallvolumes of liquid (typically 0.01 ml) as may be required for the elutionprocess.

The method and device according to the invention are especially suitablefor use with isolation procedures, where, usually an ingredient is to beisolated in rather pure form from a relatively large volume of samplefluid, and concentrated into a smaller volume of another fluid to besuitable for further use.

In the case of a method for the isolation of nucleic acid such furtheruse may be a nucleic acid amplification method or an assay for thedetection of nucleic acid or both.

A method and apparatus for separating and resuspending superparamagneticparticles is disclosed in the application WO-A-91/09308. In thisapplication it was disclosed that superparamagnetic particles may beaggregated and resuspended by subsequent application of differentmagnetic fields. First and second applications of the magnetic fieldcould be provided with the same magnet, which was then rotated aroundthe container containing the particles to a different location. Twospaced opposed electromagnets, however, could also be used. Theseelectromagnets were energized alternately to produce the first andsecond magnetic fields that keep the particles in suspension and mixthem with the fluid in which they were contained.

A method for the separation of magnetic particles from a fluid isdisclosed in U.S. Pat. No. 3,985,649. The particles may be separatedfrom a fluid by bringing the particles into close proximity with amagnet and moved through the liquid along the wall of a container. Theymay even be moved out of the liquid in this way and can be transportedto a second container.

In U.S. Pat. No. 4,988,618, a device is described for use with assayswherein multiple small volume samples are tested at the same time. Thistype of assay can be performed in, for example, microtiter plates.Magnetic microparticles are present in each well of the microtiterplate. The device thus has multiple orifices and the orifices are eachsurrounded by multiple permanent magnets, preferably four. The resultingstructure of magnets and orifices is rigid; the magnets are not intendedto be moved and are mounted in a fixed relation with respect tothemselves and to the base of the device. All magnetic are aligned andthe field orientation of the magnets may be such that all magnets havethe same field direction or neighbouring magnets have opposite fielddirections. The magnets orientation thus results in four spot attractionsites per orifice. The magnets are purely meant for separation purposes.It is disclosed in the patent that the device may further comprise meansor agitating the reagents within the containers.

The applicant has already filed an international applicationWO-A-01/05510 which proposes a solution to improve the mixing. Itrelates to a method and device, which allows efficient mixing ofmagnetic or magnetizable particles in a fluid and optionally separationof the particles from said fluid. Use is made of magnetic field ofopposite and changing directions. It has been found that, when magneticor magnetizable particles in a fluid are subjected to these magneticfields, the particles are, under the influence of the filed, efficientlycontacted with the fluid. Such particles normally may tend to form aclot, which can prevent efficient mixing with a fluid. It has been foundthat, by subjecting the container in which the fluid and the particlesare comprised, to magnetic fields of different and changing directions,the particles are efficiently separated from each other and drawn troughthe fluid in such a way that a very efficient mixing process occurs. Themethod allows efficient mixing of particles with even very small fluidvolumes. The method of the invention therefore has the advantage that itmay save in, for example, washing fluids and may allow the reduction ofthe volume of fluid needed. Thus, for example in isolation procedures,the method of the invention allows the purification of reagents in highconcentrations. Beside, whereas prior art methods can be laborious andtime consuming; the method is fast and easy to perform.

Thus, provided with this application is a method of mixing, in one ormore container(s), magnetic or (super) paramagnetic particles with afluid, using more than one magnets, whereby the containers are subjectedto magnetic fields with different and changing directions by moving themagnets with respect to the position of the container(s) and/or bymoving the containers with respect to the positions of the magnets.

State of the Art Concerning the Concentration Step:

Many diagnostic tests are carried out after steps of extracting thetarget analytes from biological samples, of purifying in order to removeparasitic products which penalize the performance of the test, ofconcentrating the target analytes in order to increase the amount ofanalyte per unit of buffer volume, and of dissolving the target analytesin a buffer in order to make them chemically accessible.

In addition, in order to increase the sensitivity and the specificity ofa test for demonstrating an analyte, it is sometimes necessary to reducethe volume of the buffer in which the copies of the analyte being soughtare found, while at the same time conserving said analyte in itsentirety.

Biologists have entirely conventional means for concentrating ananalyte, in particular using centrifugation, filtration and/or magneticsedimentation techniques. These techniques require transfers ofsolutions and manipulations of the analyte, which lead to an inevitabledecrease in the amount of analyte that can be analysed.

For example, in centrifugation and magnetic sedimentation methods, theactual centrifugation or magnetic sedimentation steps may have to berepeated several times, the limit of the number of repetitions being setby the minimum volume of solution which can be easily and reliablyhandled with a conventional pipette. This minimum volume is of the orderof about 10 micro litres. Below this, transporting it in “large”containers such as pipettes, flasks, etc loses liquid, and thereforeanalyte. In addition, there are problems of evaporation and ofadsorption to the walls of the containers during these manipulations.

In the case of a low concentration of analyte in the starting sample,this may cause the complete disappearance of the analyte or a decreasein the amount thereof such that it may become undetectable.

Besides the abovementioned drawbacks, these manipulations are expensivein terms of material and take a lot of time. This remains a constantproblem for many industrial applications, for example the detection ofpathogenic micro organisms in a biological specimen or an industrialsample.

The international application, WO-A-02/43865, concerns a method fortransporting an analyte present in a sample, a method for concentratingan analyte present in a sample, and a device for implementing saidmethods. The method for transporting an analyte present in a sampleconsists in preparing a solution from the sample wherein the analyte isfixed on magnetic particles; introducing this solution in a firstcontainer connected via a bottle-neck to a second_container; displacingwith a magnetic system the analyte fixed on magnetic particles from thefirst container to the second container via the bottle-neck; the secondcontainer being filled with all or part of said solution and/or withanother solution.

Here again, the solution is provided to how to concentrate magneticparticles into a small volume of liquid. However the separation is notefficiently treated, especially if the magnet is of a smaller sizecompared to the first container, and the mixing is even not discussed.

A real need therefore exists for a method and a device for treatinganalytes bound to magnetic particles while at the same time conservingthe amount of analyte present at the start, for example in order toincrease the sensitivity and the specificity of diagnostic tests and ofany chemical reaction directed towards the analyte, and to overcome theabovementioned drawbacks.

The present invention satisfies this need, and has not only theadvantage of overcoming the abovementioned drawbacks, but also manyother advantages, which those skilled in the art will not fail to note.

Thus none of the above-described documents presents a process offeringthe advantages of separating, mixing and concentrating molecules ofinterest from a liquid sample in only one container. Likewise, they donot propose a container having technical features authorizing therunning of such a process. Regardless of the claimed method, it may beeither manual or automatic, or operated by an automated device or not.

This is the main goal of the present invention to propose a process formanipulating magnetic particles that are suspended in a fluid, possiblycontaining a biological entity of interest, the magnetic particles beingable to bind the entity of interest, the fluid being contained in areaction vessel constituted by a large upper compartment with a funnelshape, an elongate lower compartment with a substantially constantcross-section and a closed base, consisting of:

-   -   a) subjecting the magnetic particles to two magnetic fields        applied simultaneously to separate all said magnetic particles        present in at least the upper compartment of the vessel from the        fluid,    -   b) transferring the separated magnetic particles from the upper        compartment to the elongated lower compartment,    -   c) removing the fluid from the vessel,    -   d) adding a washing liquid to the lower compartment,    -   e) subjecting the rest of the fluid to at least two magnetic        fields applied successively with different and changing        directions to wash all the magnetic particles present in the        lower compartment, and    -   f) concentrating said magnetic particles in said lower        compartment.

In a particular configuration of the invention, the two magnetic fieldsapplied simultaneously are generated by at least two magnets, the poleaxis of the magnets forming together an angle different from 180°,preferably included between 30 and 150° and more preferably includedbetween 60 and 120°.

In another particular configuration of the invention, the at least twomagnetic fields applied successively are generated by at least twomagnets, the pole axis of the magnets being parallel one to the other.

In a specific embodiment of the latter particular configurationpresented just above, the successive magnetic fields with different andchanging directions are applied to the vessel by moving the magnets withrespect to the position of the vessel and/or by moving the vessel withrespect to the position of the magnets.

Always in a particular configuration of the invention, the at least twomagnets, cooperating together and having coaxial magnetic fields, and atleast two magnets, cooperating together and having magnetic fields thatare not coaxial, are positioned on both opposite sides of the vessel.

In a particular configuration of the invention, the magnets areinterdependent with one support.

In another particular configuration of the invention, the magnetsintended to the separation and the magnets intended to the mixing areidentical.

In one embodiment, the support can be moved in rotation around (to passfrom separation to mixing configurations) and longitudinally along (torealize the mixing step) an axle passing through each magnet, the axlebeing parallel to the moving, defined previously, and perpendicularly tomagnet's pole axis.

In a particular configuration of the invention, the magnets intended tothe separation and the magnets intended to the mixing are different.

According to any of the former particular configurations of theinvention, the lower compartment is constituted by:

-   -   one medium compartment to which the magnetic fields are applied        successively for washing the magnetic particles, and    -   one bottom compartment in which the magnetic particles are        concentrated and to which the magnetic fields are applied        successively to bring the particles into contact with the        elution buffer.

In one particular configuration, in the main process, above disclosed,between step e) and step f), the following intermediate steps arerealized:

-   -   e1) transferring the separated and mixed magnetic particles from        said medium compartment to said bottom compartment,    -   e2) removing the washing liquid from the vessel, and    -   e3) adding an elution buffer to the bottom compartment.

In one particular configuration, in the main process, above disclosed,the following further steps are realized after step f):

-   -   g) transferring the magnetic particles from said bottom        compartment to the medium compartment or to the upper        compartment,    -   h) removing the elution buffer present in the bottom compartment        and containing the entity of interest for further processing.

In a preferential configuration, the volume of the medium compartment issmaller compared to the upper compartment and bigger compared to thebottom compartment.

In a particular configuration, the magnet brings about the transfer ofmagnetic particles.

According to any of the former particular configurations of theinvention, the molecules of interest are constituted by nucleic acids(RNA and/or DNA).

On the one hand, the binding of the nucleic acids is non-specific andrealized directly onto the surface of the magnetic particles.

On the other hand, the binding of the nucleic acids is specific andrealized directly onto capture probes carried by the surface of themagnetic particles.

The present invention also relates to a device for extracting possiblemolecules of interest from a fluid to which is added a suspension ofmagnetic or (super) paramagnetic particles contained in at least onereaction vessel and capable to bind the molecules of interestcomprising:

-   -   at least one separating station to capture the magnetic        particles present in a large upper compartment of each reaction        vessel,    -   at least one washing station to mix said magnetic particles        present in a medium compartment of each reaction vessel,    -   at least one concentrating station to mix said magnetic        particles present in a bottom compartment of each reaction        vessel, and    -   at least one pipetting means to dispense and/or to remove part        or all of the fluid, the washing liquid and output buffer that        are needed to support the process as above presented.

In one embodiment of the device, the separating station comprises atleast two magnets, the pole axis of the magnets forming together anangle different from 180°, preferably included between 60 and 150° andmore preferably included between 80 and 120°.

In another embodiment of the device, the washing station comprises atleast two magnets, the pole axis of the magnets being parallel one tothe other.

The invention also relates to a reaction vessel that can be used in anextracting device, above exposed, comprising:

-   -   a top aperture,    -   one upper compartment with a funnel shape,    -   one medium compartment with a substantially constant        cross-section,    -   one bottom compartment with a substantially constant        cross-section,    -   a closed base, and    -   a longitudinal axis defined by the lower compartment.

According to one embodiment of the reaction vessel, each of the oppositewalls of the upper compartment, constituting the funnel shape, isperpendicular to the pole axis of the magnets that is present withrespect to this wall.

According to another embodiment of the reaction vessel, the oppositewalls of the upper compartment form together an angle different from180°, preferably included between 60 and 150° and more preferablyincluded between 80 and 120°.

According to any of the embodiment of the reaction vessel abovedescribed, the volume of the medium compartment is smaller compared tothe upper compartment (and bigger compared to the bottom compartment.

Again according to any of the embodiment of the reaction vessel abovedescribed, the ratio between the volumes of the medium compartment andthe upper compartment or between the volumes of the bottom compartmentand the medium compartment is comprised between 1:2 to 1:100, preferablybetween 1:5 to 1:20, and more preferably 1:10.

The invention also relates to a set of reaction vessels constituted byat least two vessels, preferentially at least five vessels and morepreferentially eight vessels, according to any of vessels presentedabove, said vessels being arranged symmetrically along one line.

According to one embodiment of the set of reaction vessels, itcooperates with at least two tips, preferentially at least five tips andmore preferentially eight tips, said tips constituting:

-   -   a first pipetting mean to dispense and/or to remove part or all        of the fluid,    -   a second pipetting mean to dispense and/or to remove part or all        of the wash liquid, and    -   a third pipetting mean to dispense and/or to remove part or all        of output buffer.

According to one embodiment of the set, the free ends of the tipsconstituting the first or the second or the third pipetting mean beingarranged symmetrically along one line or two lines, the two lines ofeach arrangement being parallel one to the other.

According to another embodiment of the set, the free ends of:

-   -   the tips constituting the first pipetting mean being arranged        symmetrically along one line,    -   the tips constituting the second pipetting mean being arranged        symmetrically along one or two lines, and    -   the tips constituting the third pipetting mean being arranged        symmetrically along two lines.

According to one embodiment of the set, the first pipetting mean, thesecond pipetting mean and the third pipetting mean constitute oneunique, two or even three different pipetting means.

With “mixing” in this context is meant that the particles and the fluidare brought in close contact. The word “mixing” thus means contacting ina very efficient manner, such as when particles would be washed orreacted with components present in the fluid. Mixing, in this context,does not necessarily provide a homogeneous mixture after the process isfinished. The particles may, when the magnets are removed, segregate tothe bottom of the container in which they are comprised or may be heldto the wall of the container in a particular location by the magnets.The mixing process can for example be used to wash the particles or toreact the particles with a component of the liquid, or to bind acomponent of the liquid to a reagent coated on the particles. Likewise,the mixing process may result in the elution of a certain componentoriginally present on the particles into the surrounding liquid. Themethod of the invention is applicable in each of these processes andprovides an efficient, rapid and convenient way of contacting magneticor magnetizable particles with a volume of a certain fluid.

The invention will now be described further, by way of examples, withreference to the accompanying drawings, in which:

FIG. 1 is a perspective view of a set constituted by eight containersaccording to the invention.

FIG. 2 is a cross-sectional view of the container set according to A-Aof FIG. 1.

FIG. 3 is a cross-sectional view of the container set according to B-Bof FIG. 2.

FIG. 4 represents detail C of FIG. 3.

FIG. 5 represents a view, which is similar to FIG. 2, in the case thevessel filled up with the biological sample is disposed between twomagnets, acting simultaneously on the fluid.

FIG. 6 represents a view, which is identical to FIG. 5, after the twomagnets have attracted all magnetic particles present in the startingsample.

FIG. 7 represents a view, which is similar to FIG. 6, when said twomagnets are moved in downward direction and transfer the magneticparticles from incubation compartment to wash compartment.

FIG. 8 represents a view, which is similar to FIG. 7, when the magnetshave reached their final position and all magnetic particles have formeda single pellet.

FIG. 9 is a cross-sectional view of one reaction vessel according to D-Dof FIG. 5.

FIG. 10 represents a view, which is similar to FIG. 8, when the vesselis disposed between two other magnets, and after withdrawal of theliquid part of the biological sample.

FIG. 11 represents a view, which is similar to FIG. 10, when the washliquid is added by way of an oblique tip to avoid splashing.

FIG. 12 represents a view, which is similar to FIG. 11, showing the toand fro movement of the magnetic particles inside the wash liquid.

FIG. 13 is a cross-sectional view of one reaction vessel according toE-E of FIG. 12 explaining the link between the to and fro movement ofthe magnetic particles and the relative movement of the set of vesselswith respect to magnets.

FIG. 14 represents a view, which is similar to FIG. 10, when the vesselis disposed between the same two magnets, but after removal of said washliquid.

FIG. 15 represents a view, which is similar to FIG. 14 concerning themagnets position, when the output or elution buffer is added by way ofthe tip, disclosed in FIG. 11, or by way of another tip.

FIG. 16 represents a view, which is quite similar to FIG. 7, when thetwo other magnets are moved downwardly and transfer the magneticparticles from wash compartment to output compartment.

FIG. 17 represents a view, which is similar to FIG. 16, after removal ofoutput buffer present in the wash compartment. At this stage, the outputbuffer is submitted to a heating system in order to achieve elution ofmolecule of interest.

FIG. 18 represents a view, which is similar to FIG. 13, explaining theto and fro movement of the magnetic particles in the output buffer dueto the relative movement of the set of vessels with respect to themagnets.

FIG. 19 represents a view, which is quite similar to FIG. 16, but usingthe two former magnets to move upwardly and transfer the magneticparticles from output compartment to wash compartment where no liquid ispresent.

FIG. 20 represents a view, which is quite similar to FIG. 19, where theupward movement is maintained so that the magnets transfer the magneticparticles from wash compartment to incubation compartment to authorizethe removal of the rest of output buffer containing the eluted moleculesof interest from the output compartment.

FIG. 21 is a perspective view of a main body of tips' support separatedfrom eight tips, tips that could be connected to tip supports having acomplementary shape.

FIG. 22 is a bottom view of a main body to which the tips have beenconnected.

Finally FIG. 23 shows a graphical presentation of the result of a set ofreal time PCR reactions where a DNA fragment of the Porcine herpesvirus(PhHV-1) has been amplified after isolating the virus from differentsamples using the device disclosed in this patent application. Along thehorizontal axis different sample numbers are indicated. The verticalaxis shows the threshold cycle for each sample when a positive PCRsignal is observed. Four different sample types have been used (CSF,plasma, serum and blood). With each sample type 20 individual sampleshave been used.

1—DESCRIPTION OF THE PREFERRED EMBODIMENT

1.1—Details of the Reaction Vessel:

The vessel 3 is well disclosed on FIGS. 1-4. It consists in threecompartments:

a large upper incubation compartment 4 with a funnel shape of the vessel3,

an elongated medium wash compartment 5 with a constant cross-section ofthe vessel 3

an elongated bottom output compartment 6 with a constant cross-sectionof the vessel 3

This configuration obliges to have a top aperture 19 where it ispossible to introduce/dispense or remove any liquid present inside saidvessel 3, but also a closed base 7. Finally, according to FIG. 4, thevessel 3 has a longitudinal axis 8.

Now referring more particularly to FIG. 1, vessels 3 in a preferentialconfiguration are arranged to constitute a set 23, having a manipulationtongue 24 that facilitates the handling by the users. Positioning meansnot represented on the Figures, could be added in order to render theinstallation in an automatic device even more efficient than usual.

The main features are:

-   -   Preferably, the total height of the vessel 3 is 40 mm to        facilitate recovery of the output buffer 21 from the output        compartment 6 using a standard disposable filter tip, like 22,        25, 26.    -   Volume of incubation compartment 4 is 2-6 ml, preferably 4 ml.        In the later configuration, which is the current design, the        dimensions are 20 mm in high, 25 mm in depth, 9 mm in width.    -   Volume of wash compartment 5 is 0.1-1 ml, preferably 0.2 ml. In        the later configuration, which is the current design, the        cylindrical shape has 5 mm as diameter and 8 mm as height.    -   Volume of output compartment 6 is 10-100 μl, preferably 20 μl.        In the later configuration, which is the current design, the        cylinder is 5 mm in height and 2 mm in diameter.        The thickness of the wall of the vessel is between 0.2 mm and 1        mm (current design: 0.5 mm).

Preferably, the reaction vessel is a plastic disposable produced byinjection moulding for example using polypropylene. To allow convenientmanipulation by an operator, it is recommended to integrate severalreaction vessels 3 into a set 23. The applicant has realized a designwhere a set comprises eight vessels 3. For the same reason it isconvenient to integrate the tips 21, 25, 26 with their common base 29 toobtain a single unit where the number of tips equals the number ofreaction vessels 3 and where the spacing between tips matches with theposition of vessels in a set 23.

Preferably the device uses magnets fabricated from neodymium (NdFeB)with a remnant field of 1.2 Tesla. With the current device, cylindricalmagnets of 6 mm diameter and 7 mm length have shown suitable for themagnets 9, 10 13 and 14.

When separating magnetic particles 2 from the liquid 1 in the incubationcompartment 4, the spacing between the face of the magnets 9 and 10 andthe wall of the reaction vessel is typically 0.5 mm. During the washmovement (F11, FIG. 13) the magnets 13 and 14 are translated withrespect to the vessel with a typical velocity of 1 cm per second. Whenpassing the vessel, the distance to the wall of the wash compartment 5is typically 1 mm. During the elution movement (F11, FIG. 18) themagnets 13 and 14 are translated with respect to the vessel with atypical velocity of 1 cm per second. When passing the vessel, thedistance to the wall of the output compartment 6 is typically 2 mm.

1.2—Method According to the Invention:

In a preferred embodiment, the device disclosed in this application iscombined with some standard components such as a suction pump and liquidline for aspirating liquid, a liquid dispenser module and somemechanical means for translating the magnets, the aspiration tip and thedispenser with respect to the reaction vessels. In a preferredembodiment, this device is useful for isolating nucleic acids from acomplex starting biological sample, such as whole blood, blood serum,buffy coat (the crusta phlogistica or leukocyte fraction of blood),urine, feces, liquor cerebrospinalis, sperm, saliva, tissues, cellcultures and the like. Nucleic acid as isolated from above-mentionedbiological material can also comprise the endogenous nucleic acid fromthe organism from which the sample is derived and any foreign (viral,fungal, bacterial or parasitic) nucleic acid.

The device is used according to the following procedure:

(a) Add Liquid Mixture to the Reaction Vessel

A mixture 1 of the primary sample, a lysis buffer and magnetic particles2 is added to the reaction vessel 3. The target molecules (nucleic acid,not shown on the Figures) are released from the cells or organisms inthe sample and bind to the magnetic particles 2. This process step isdefined as “incubation”. In this step, all the compartments,constituting said vessel 3, are filled up with the fluid 1, i.e.incubation compartment 4, wash compartment 5 and output compartment 6.Due to the difference in size in between said compartments 4-6, most ofsaid fluid 1 is contained in the incubation compartment 4 of thereaction vessel 3. Typically, the incubation time is about 5 minutes.

(b) Separate Particles From the Sample

After incubation, the liquid mixture 1 in the vessel 3 is disposedbetween two magnets 9A or 9B or 9C, etc. and respectively 10A or 10B or10C, etc.; each one 9 or 10 generating a magnetic field respectively 1and 12, as disclosed in FIG. 5. The configuration is also well definedin FIG. 9. As a result, the magnetic particles 2 are collected—as apellet—at two positions at the sidewall of the incubation compartment 4corresponding to the location of the magnets (FIG. 6). Collection timeis typically around 1 minute.

(c) Transfer Particles to Wash Compartment

According to FIG. 7, the pellets of magnetic particles 2 are transferredfrom the incubation compartment 4 to the wash compartment 5 by movingthe magnets 9 and 10 in the downward direction according to F2. It isalso possible to achieve this movement by moving the vessel 3 in theupward direction according to F1, or to combine the down (F2) and upward(F1) movements of respectively said magnets 9 and 10 and said vessel 3.During that movement(s), the particles 2 initially collected by magnet10, jump over towards magnet 9, according to F3, where they join withthe particles 2 initially collected by magnet 9. After thisconsolidation, all magnetic particles 2 are transferred to the washcompartment 5 as shown in FIG. 8.

(d) Wash Magnetic Particles

The magnetic particles 2 are washed to remove all sample components aswell as other reagents that could interfere with the downstreamapplication. Washing is achieved by disposing the reaction vessel 3 tothe magnets 13 and 14 and subsequent removal (F5) of the liquid samplefrom the reaction vessel 3 using the tip 22 (FIG. 10), which isintroduced vertically, according to F4, in said vessel 3. Next,according to FIG. 11, a fresh wash liquid 20 is added into said vessel 3according to F9 by mean of a tip 25 introduced in the vessel accordingto F8, which is at an oblique angle compared to the vertical position ofthe vessel 3 in order to eliminate any risk of splashes that couldgenerate drops on the internal sidewall, prejudicial to further futureprocess steps. During sample removal and liquid addition, the magneticparticles 2 are retained at the sidewall of the wash compartment 5 underthe action of the magnets 13 and 14. Next, the magnets 13 and 14 aremoved in the direction F11, as depicted on FIG. 13, to transfer theparticles 2 back and forth, according to F10 of FIG. 12, betweenopposite sides of the wash compartment 5 to bring them in contact withthe wash liquid 20. In those circumstances, magnetic particles 2 arealternatively submitted to two opposite magnetic fields 15 and 16.

Obviously it is also possible to achieve this movement in the F11direction by moving the vessel 3 with respect to the magnets 13 and 14,or to combine movements of both said magnets 13 and 14 and said vessel3.

Step (d) can be repeated until sufficient wash performance has beenobtained. If appropriate a sequence of different wash liquids can beused. In case different wash liquids are used it will be clear that thedispense tip is washed adequately when changing buffers.

The magnets 13 and 14 are placed in intervening array geometries. Thislayout allows the use of the method of the invention to give a highthroughput format. An embodiment wherein the vessels and the magnets areplaced in intervening array geometries is illustrated in FIG. 13. Thevessels 3 are placed in array geometry with the magnets 13A, 13B, 13C,etc., on one side and 14A, 14B, 14C, etc., on the other side, fixed to asecond array 18, used as a support for the magnets, that translates withrespect to the vessels 3.

In this way a large series of samples is processed simultaneously.Addition and aspiration of liquids may be by hand or by an automatedmulti-tip dispenser instrument as know in the art. Such a configurationis well exposed by the Applicant in a former application filed underapplication number WO-A-01/05510 published the Jan. 25, 2001 andentitled: “Device and method for mixing magnetic particles with a fluidand optionally separating the particles from the fluid and use thereofin purification methods”. Readers could find relevant information aboutsaid configuration in this document. This is the reason why its contentis enclosed for reference.

(e) Prepare for Elution

While the magnetic particles 2 are retained at the sidewall of the washcompartment 5, all wash liquid is removed from the vessel 3, accordingto F13, using tip 26 that is introduced in the vessel 3 according toF12, to end up in the configuration as depicted in FIG. 14. Next, anoutput or elution buffer 21 is added by way of tip 26 or a new tip, notrepresented on the drawing, introduced according to F14, to the reactionvessel 3 to fill (F15) the output—and the wash compartments 6 and 5.After that, the magnetic particles 2 are transferred from the washcompartment 5 to the output compartment 6 by moving the magnets 13 and14 in the downward direction (F16) and/or by moving the vessels 3 in theupward direction (F17) (FIG. 16). Next, the volume of output buffer 21is set by descending tip 26 into the reaction vessel 3, according toF18, while aspirating part of the liquid, according to F19, to a levelthat corresponds with the specified volume of output buffer (FIG. 17).

Although the magnetic particles 2 are in contact with the elution buffer21 no significant release of target takes place due to the fact that:

-   -   the contact time is short,    -   said magnetic particles 2 are collected as a clump, and    -   the elution buffer 21 is at room temperature.

(f) Heat the Elution Buffer

A heater element 33 is enclosing the output compartment 6 of thereaction vessel 3 to heat the output buffer to a specified temperature(FIG. 17). A typical temperature for the elution process is 60-80° C.This temperature is applied during about 15 seconds to 10 minutes,preferably 1 minute to 6 minutes and more preferably 3 minutes. Theseconditions authorize significant release of target.

(g) Elute Nucleic Acid From Magnetic Particles

The target molecules are released from the magnetic particles 2 andrecovered in the output buffer 21, only present in the bottomcompartment 6, by moving the magnets 13 and 14 in the direction F11,according to FIG. 18. Similar to the washing step (d), the particles 2are translated back and forth between opposite sides of the outputcompartment 6 to bring said particles 2 in efficient contact with theoutput buffer 21. During this step the heater 33 is enclosing the outputcompartment 6 to control the temperature of the output buffer 21.

(h) Separate Magnetic Particles From Output Buffer

After the elution step, the magnetic particles 2 are cleared (separated)from the output buffer 21 by disposing the sample vessel 3 to themagnets 9 and 10, see FIG. 19, to allow magnet 9 to collect theparticles 2 at the sidewall of the output compartment 6 and subsequentlymove the vessel 3 in the downward direction, according to F20, and/ormove the magnets 9 and 10 in the upward direction, according to F21, inorder to maintain said particles 2 at the sidewall of the incubationcompartment 4, as disclosed in FIG. 20, or optionally of the washcompartment 5.

Then the elution buffer 21 present in the output compartment 6 could beused for further processing or, according to FIG. 20, be transferred bymean of tip 26, which is introduced (F22) into the vessel 3 and theelution buffer 21, containing the target molecules, is removed byaspiration (F23).

It will be clear to anybody skilled in the art that the device disclosedabove as well as the method for using the device is easily integrated inan automated system by performing the translations for the reactionvessel, the magnets, the heater and the tips in an automated way, forexample using a set of linear actuators controlled by stepper motors. Itis also obvious that, depending on the particular application,particular steps of the above protocol can be omitted such as (f), (g)or (h).

FIGS. 10 and 14 are quite similar, as the magnetic particles 2 aremaintained in position in a single vessel by a only one magnet 13, forinstance. Each neighbouring vessel 3 of the set 23 contains alsomagnetic particles 2 that are attracted by magnet 14. In other words,neighbouring vessels 3 have their magnetic particles 2 on oppositesidewalls. To facilitate the introduction of tips 22 or 25 in the eightvessel's set 23, the positioning of said eight tips has been implementedin an improved manner, as disclosed in FIGS. 21 and 22. Thus all thetips that collaborate with one set 23 of vessels 3 are associated with amain body 29, this latter having eight tip supports 30, each 30 shiftedone to the other. More specifically, each tip comprises two free ends,one is the lower end 27 and the other one is the upper end 28 of thetip. The free ends 27 or 28 are arranged symmetrically along two lines,each line being drawn by four lower end 27 or four upper end 28, and thetwo lines being parallel one to the other.

Obviously when the tips 22 or 25 are introduced in the vessels 3,according to FIGS. 10 and 14, the tips are taken away as much aspossible from the magnetic particles 2, to avoid any contact prejudicialto further future process steps.

Now referring more particularly to FIG. 21, main body 29 in apreferential configuration comprises a manipulation tongue 32 thatfacilitates the handling by the users. Positioning means notspecifically represented on the Figures, could be added in order torender the installation in an automatic device even more efficient thanusual. Moreover, said main body 29 comprises an admission/exhaustaperture 31 to permit the dispensing or the removing of any fluid. Thisaperture 31 could also serve as positioning means.

Four examples are presented where nucleic acids are isolated from aninput sample using the invention presented above. With these examplesthe wash buffers, the elution buffer and the magnetic particles wereobtained from the “NucliSens magnetic extraction reagents” (article code200297). The lysis buffer is the “NucliSens Lysis buffer” (article code200295). NucliSens products are supplied by bioMérieux B. V. (Boxtel,The Netherlands)

2—EXAMPLE 1 Concentration of Ribosomal RNA From Plasma Samples

2.1—Materials and Methods:

Target RNA is extracted from Escherichia coli cells using the QiagenRNA/DNA maxi kit (article code 14162, Qiagen, Hilden, Germany). For eachsample, 2 micrograms (μg) are used as input.

Sample is constituted by normal EDTA/citrate plasma samples from a poolof 100 individual blood donations.

The RNA quantification is performed using a luminescent marker for RNA(Sybr Green-II, supplied by Molecular Probes: article S-7564) incombination with a luminescent reader for microtiter plates (“Victor²”,supplier Wallac Oy, Turku, Finland: 1420 multilabel counter).

Purity of the output buffer is determined from the relative value forthe spectral absorption at 260 nm and 280 nm (A260/A280) using an UVspectrophotometer (supplier Unicam, Cambridge, Great Britain: UnicamUV-1).

2.2—Sample Preparation:

A set of twenty-four plasma samples is prepared as follows:

-   -   With each sample, 1 ml of plasma is added to the reaction vessel        and mixed with 2 ml NucliSens Lysis buffer.    -   Next, the target rRNA is added to that mixture using an input of        2 μg RNA for each sample.    -   Subsequently, 1 mg of magnetic particles is added to the lysed        plasma samples and a homogeneous dispersion is produced using a        manual pipette.

2.3—Extraction Method:

A batch of twenty-four samples is processed simultaneously according tothe procedure indicated above as steps (a) to (g). The samples are addedto three sets of reaction vessels, each set comprising eight identicalvessels.

After collecting the magnetic particles from the incubation compartment(collection time was 1 minute), the particles are transferred to thewash compartment and washed using NucliSens wash buffer 1 and washbuffer 2, using two cycles of 3 ml and 1 ml in the first and secondcycle respectively.

After washing, the particles are transferred to the output compartmentwhere the target RNA is released from the particles by mixing theparticles in the NucliSens elution buffer (wash buffer 3) for 5 minutes.During elution, the temperature of the buffer is 60° C. The volume ofthe output buffer is 20 μl.

After elution, the magnetic particles are separated from the outputbuffer using the magnets 9.

After completing the protocol, the amount of RNA recovered in the outputcompartment of each vessel is measured using a luminescent RNA marker.For that purpose, 10 μl of the output buffer is transferred from eachreaction vessel into the well of a microtiter plate and 190 μl of SybrGreen solution is added to each well. In each well, the optical signaldetected by the luminescent plate reader (Victor² is a direct measurefor the amount of RNA that is present in that well.

2.4—Result:

For the twenty-four samples, the average amount of target rRNA in theoutput compartment of the vessel is 1.32 μg with a standard deviation of50 ng. This corresponds to an average yield of 66%.

Purity of the output is excellent as concluded from the A260/A280 ratiousing the UV spectrophotometer. The output buffer shows a ratio of 2.1whereas pure RNA in this buffer has an A26/A280 ratio between 1.9 and2.1.

Consistency of the target RNA is determined using the 2100 Bioanalyzer(Agilent Technologies, Amstelveen, the Netherlands). This instrumentdetects the two discrete RNA bands corresponding to the 16S and 23S RNA.No other products such as smaller RNA fragments have been detected.

The total time for completing the extraction procedure for this batch oftwenty-four samples is 30 minutes, starting after adding the magneticparticles to the lysed samples. No intervention from an operator isneeded to produce the concentrated RNA in the output.

3—Example 2 Concentration of Plasmid DNA From Plasma Samples

3.1—Materials and Methods:

Target is a plasmid DNA, pBR322 (article N3033L, New England BiolabsInc., Beverly, Mass.), linearized by BamH1 (article E1010WH, AmershamBioscience Corp, NY, USA). Six μg of DNA is used as input per sample

The sample is constituted by 0.1 ml normal EDTA/citrate plasma fromblood donations

3.2—Sample Preparation:

Twenty-four samples are processed simultaneously, using the sameprocedure as with example 1. The individual plasma samples are added tothe reaction vessels. Next, 2 ml of NucliSens lysis buffer are added.Subsequently, the plasmid DNA is spiked to the sample and 1 mg ofmagnetic particles is added to the mixture.

3.3—Extraction Method:

The collection step, washing steps and elution step of the extractionprocedure are identical to example 1.

3.4—Result:

After completing the procedure, the amount of extracted DNA isdetermined from the optical density of the output buffer at 260 nm.

The average amount of DNA recovered from the reaction vessel is 3.1 μgwith a variation coefficient of 4% between different vessels. Thiscorresponds to an isolation yield of 52% using 6 μg of DNA as input.

The average A260/A280 ratio for the output buffer is 1.9. For pure DNAin the output buffer this ratio is between 1.7 and 2.1.

As with example 1, the time needed to complete the extraction procedureis 30 minutes starting after adding the magnetic particles.

4—EXAMPLE 3 Recovery of Viral RNA From Sputum Samples

4.1—Materials and Methods:

In this experiment, the recovery of HIV RNA that is spiked to ninesputum samples is determined using the NucliSens EasyQ HIV-1 assayversion 1.1 (article 285029, bioMérieux B.V., Boxtel, The Netherlands).

The RNA is HIV RNA obtained from a cultured HIV-1 type B virus stock(HXB2) that is lysed using the NucliSens lysis buffer and calibratedagainst the WHO International HIV-1 RNA standard.

The sputum samples were obtained from the University Hospital ofAntwerpen (Belgium).

4.2—Sample Preparation:

With each sputum sample a volume of 1 ml is mixed with 0.5 ml ofprotease solution (100 mg/ml) and incubated on a plate shaker for 20minutes at room temperature to obtain a liquefied sample.

The target HIV RNA is spiked to a tube containing 2 ml of NucliSenslysis buffer, using 30,000 copies input for each sample. Together withthe HIV RNA, a calibrator RNA that is part of the EasyQ kit is added tothe lysis buffer. Next the liquefied sputum samples are added to thereaction vessel together with the lysis buffer (2 ml) containing theRNA. After 10 minutes, 1 mg of magnetic particles is added to eachsample. A homogeneous dispersion is obtained by mixing with a manualpipette.

4.3—RNA Extraction:

The extraction procedure proceeds as with the examples 1 and 2 describedabove.

4.4—RNA Detection:

At the end of the extraction procedure, the HIV RNA and calibrator RNAare concentrated in 12 μl of elution buffer in the output compartment.Two fractions of 5 μl each are aspirated from the output compartment andused as input in a NucliSens EasyQ assay. For the detection, theprocedure as indicated in the EasyQ assay manual is followed.

4.5—Result:

The results are presented in the table here below: EasyQ result (viralcopies) Sample number Reaction 1 Reaction 2 1 11.000 10.000 2 11.0009.700 3 9.600 9.200 4 12.000 13.000 5 11.000 16.000 6 18.000 13.000 79.200 10.000 8 12.000 14.000 9 20.000 12.000

From these results, we conclude that the device disclosed in thisapplication is well able to isolate viral RNA from sputum samples andconcentrate it in a form that facilitates detection using standardamplification methods.

5Experiment 4: Recovery of Viral DNA From Different Sample Types

5.1—Materials and Methods:

This experiment represents a series of four extraction runs ontwenty-two samples each.

With each run a different sample type is used:

-   -   Run 1: plasma samples from individual donations (1 ml per        sample)    -   Run 2: Cerebra Spinal Fluid (CSF) (0.1 ml per sample)    -   Run 3: serum samples from individual donations using 1 ml per        sample    -   Run 4: whole blood samples (0.1 ml per sample).

5.2—Sample Preparation:

Each sample is added to 2 ml NucliSens lysis buffer.

As a target, the Porcine herpesvirus (PhHV-1) is spiked to twenty of thelysed samples in each run. Two samples (21^(st) and 22^(nd)) were usedas a negative control (no DNA spiked).

5.3—DNA Extraction:

The extraction method for each run (sample type) is identical to theprocedure described in the examples 1, 2 and 3.

5.4—DNA Detection:

The DNA that is recovered in the output buffer is detected using a realtime PCR. The number of PCR cycles (CT) needed to produce a positivesignal is determined for each sample.

The FIG. 23 shows a graphical presentation of these CT values for allsamples. The negative control samples were all negative.

5.5—Result:

From this set of experiments, we conclude that the device disclosed inthis patent application is well able to isolate nucleic acid from virusparticles from different sample types with excellent yield.

REFERENCES

-   1. Fluid or mixture-   2. Magnetic particles-   3. Reaction vessel-   4. Large upper incubation compartment with a funnel shape of the    vessel 3-   5. Elongate medium wash compartment with a constant cross-section of    the vessel 3-   6. Elongate bottom output compartment with a constant cross-section    of the vessel 3-   7. Closed base of the vessel 3-   8. Longitudinal axis of the vessel 3-   9. Magnets (A, B, C, D, etc.) present on one side of the container    set used for the separation step-   10. Magnets (A, B, C, D, etc.) present on the other side of the    container set used for the separation step-   11. Pole axis (A, B, C, D, etc.) of the magnet (9A, 9B, 9C, 9D,    etc.)-   12. Pole axis (A, B, C, D, etc.) of the magnet (10A, 10B, 10C, 10D,    etc.)-   13. Magnets (A, B, etc.) present on one side of the container set    used for the mixing and/or the concentrating step(s)-   14. Magnets (A, B, etc.) present on the other side of the container    set used for the mixing and/or the concentrating step(s)-   15. Pole axis (A, B, etc.) of the magnet (13A, 13B, 13C, 13D, etc.)-   16. Pole axis (A, B, etc.) of the magnet (14A, 14B, 14C, 14D, etc.)-   17. Support of the magnets (9A, 9B, 9C, 9D, etc., or 10A, 10B, 10C,    10D, etc.)-   18. Support ofthe magnets (13A, 13B, 13C, 13D, etc., or 14A, 14B,    14C, 14D, etc.)-   19. Top aperture of the vessel 3-   20. Wash liquid-   21. Output buffer-   22. Tip for adding or removing fluid 1-   23. Set of vessels 3-   24. Manipulation tongue of vessels' set 23-   25. Tip for adding wash liquid 20 and/or elution buffer 21-   26. Tip for removing wash liquid 20 and/or output buffer 21-   27. Lower end of tip 25-   28. Upper end of tip 25-   29. Main body of tips' support 30-   30. Tip support-   31. Admission/exhaust aperture of the main body 29-   32. Manipulation tongue of main body 29-   33. Heating system-   F1. Upward movement of vessel 3-   F2. Downward movement of magnets 9 and 10-   F3. Magnetic attraction of particles 2-   F4. Downward movement of tip 22-   F5. Removing of fluid 1 by aspiration with tip 22-   F8. Downward movement of tip 25-   F9. Dispensing wash liquid 20 expulsed from tip 25-   F10. Movement to and fro of magnetic particles 2-   F11. Relative movement of the set 23 with respect to magnets 13 and    14-   F12. Downward movement of tip 25-   F13. Removing of wash liquid 20 by aspiration with tip 25-   F14. Downward movement of tip 26-   F15. Dispensing output buffer 21 expulsed from tip 26-   F16. Downward movement of magnets 13 and 14-   F17. Upward movement of vessel 3-   F18. Downward movement of tip 26-   F19. Removing output buffer 21 present in wash compartment 5-   F20. Downward movement of vessel 3-   F21. Upward movement of the magnets 13 and 14-   F22. Downward movement of tip 26-   F23. Removing of output buffer 21 by aspiration with tip 26

1. A process for manipulating magnetic particles that are suspended in afluid, the magnetic particles being able to bind an entity of interest,the fluid being contained in a reaction vessel constituted by a largeupper compartment with a funnel shape, an elongate lower compartmentwith a substantially constant cross-section and a closed base, theprocess comprising: a) subjecting the magnetic particles to two magneticfields applied simultaneously to separate all said magnetic particlespresent in at least the upper compartment of the vessel from the fluid,b) transferring the separated magnetic particles from the uppercompartment to the elongated lower compartment, c) removing the fluidfrom the vessel, d) adding a washing liquid to the lower compartment, e)subjecting the rest of the fluid to at least two magnetic fields appliedsuccessively with different and changing directions to wash all themagnetic particles present in the lower compartment, and f)concentrating said magnetic particles in said lower compartment.
 2. Aprocess according to claim 1, wherein the two magnetic fields appliedsimultaneously are generated by at least two magnets, the pole axis ofthe magnets forming together an angle different from 180°.
 3. A processaccording to any of claim 1, wherein the at least two magnetic fieldsapplied successively are generated by at least two magnets, the poleaxis of the magnets being parallel one to the other.
 4. A processaccording to claim 3, wherein the successive magnetic fields withdifferent and changing directions are applied to the vessel by movingthe magnets with respect to position of the vessel and/or by moving thevessel with respect to the position of the magnets.
 5. A processaccording to claim 1, wherein the subjecting the magnetic particle totwo magnetic fields is carried out using at least two magnets, with atleast one magnet positioned on an opposing side of the vessel from atleast one other magnet.
 6. A process according to claim 5, wherein themagnets are interdependent with one support.
 7. A process according toclaim 1, wherein the subjecting the magnetic particle to two magneticfields is carried out using at least two magnets, and wherein themagnets intended to the separation are also used to mix the fluid in thevessel.
 8. A process according to claim 7, wherein the magnets areinterdependent with one support, and wherein the support can be moved inrotation around, to pass from separation to mixing configurations, andlongitudinally along, to realize the mixing step, an axle passingthrough each magnet, the axle being parallel to moving of the vesselwith respect to the magnets and/or the magnets with respect to thevessel, and perpendicularly to magnet's pole axis.
 9. A processaccording to claim 1, wherein the subjecting the magnetic particle totwo magnetic fields is carried out using at least two magnets, andwherein magnets configured to separate the magnetic particles aredifferent from magnets configured to mix the fluid in the vessel.
 10. Aprocess according to claim 1, wherein the lower compartment isconstituted by: one medium compartment to which the magnetic fields areapplied successively, and one bottom compartment in which the magneticparticles are concentrated.
 11. A process according to claim 10, whereinbetween step e) and step f) of claim 1 the following intermediate stepsare realized, and wherein the washing in step e) comprises mixing themagnetic particles in the fluid: e1) transferring the separated andmixed magnetic particles from said medium compartment to said bottomcompartment, e2) removing the washing liquid from the vessel, and e3)adding an elution buffer to the bottom compartment.
 12. A processaccording to claim 11, wherein the following further steps are realizedafter step f) of claim 1: g) transferring the magnetic particles fromsaid bottom compartment to the medium compartment or to the uppercompartment, h) removing the elution buffer present in the bottomcompartment and containing the entity of interest for furtherprocessing.
 13. A process according to claim 10, wherein the volume ofthe medium compartment is smaller compared to the upper compartment andbigger compared to the bottom compartment.
 14. A process according toclaim 1, wherein the transfer of magnetic particles is sustained by themagnet.
 15. A process according to claim 1, wherein the entity ofinterest comprises molecules constituted by nucleic acids (RNA and/orDNA).
 16. A process according to claim 15, wherein the binding of thenucleic acids is non-specific and realized directly onto the surface ofthe magnetic particles.
 17. A process according to claim 15, wherein thebinding of the nucleic acids is specific and realized directly ontocapture probes carried by the surface of the magnetic particles. 18.Device for extracting possible molecules of interest from a fluid towhich is added a suspension of magnetic or (super) paramagneticparticles contained in at least one reaction vessel and capable to bindthe molecules of interest comprising: at least one separating station tocapture the magnetic particles present in a large upper compartment ofeach reaction vessel, at least one washing station to mix said magneticparticles present in a medium compartment of each reaction vessel, atleast one concentrating station to mix said magnetic particles presentin a bottom compartment of each reaction vessel, and at least onepipetting device to dispense and/or to remove part or all of the fluid,the washing liquid and output buffer used in an extraction process. 19.Device according to claim 18, characterized in that the at least oneseparating station comprises at least two magnets, a pole axis of themagnets forming together an angle different from 180°.
 20. Deviceaccording to claim 18, characterized in that the at least one washingstation comprises at last two magnets, a pole axis of the magnets beingparallel one to the other.
 21. Reaction vessel adapted for use in thedevice of claim 18, comprising: top aperture, one upper compartment witha funnel shape, one medium compartment with a substantially constantcross-section, one bottom compartment with a substantially constantcross-section, a closed base, and a longitudinal axis defined by thelower compartment.
 22. Reaction vessel according to claim 21, whereinthe at least one separating station and the at least one washing stationcooperate with at least two magnets, each having a pole axis, andwherein each of the opposite walls of the upper compartment,constituting the funnel shape, is perpendicular to the pole axis of themagnets that is present with respect to this wall.
 23. Reaction vesselaccording to claim 22, characterized in that the opposite walls of theupper compartment form together an angle different from 180°. 24.Reaction vessel according to claim 21, wherein the volume of the mediumcompartment is smaller compared to the upper compartment and biggercompared to the bottom compartment.
 25. Reaction vessel according toclaim 21, wherein each compartment has an associated volume and whereina ratio between the volumes of the medium compartment and the uppercompartment or between the volumes of the bottom compartment and themedium compartment is between 1:2 to 1:100.
 26. Set of reaction vesselsconstituted by at least two vessels, said vessels being arrangedsymmetrically along one line.
 27. Set of reaction vessels according toclaim 26, wherein the set of vessels cooperates with at least two tips,said tips constituting: a first pipetting device to dispense and/or toremove part or all of the fluid, a second pipetting device to dispenseand/or to remove part or all of the washing liquid, and a thirdpipetting device to dispense and/or to remove part or all of outputbuffer.
 28. Set of reaction vessels according to claim 27, wherein thetips have free ends, and wherein the free ends of the tips constitutingthe first, the second or the third pipetting device being arrangedsymmetrically along one line or two lines, the two lines of eacharrangement being parallel one to the other.
 29. Set of reaction vesselsaccording to claim 28, wherein the free ends of: the tips constitutingthe first pipetting device being arranged symmetrically along one line,the tips constituting the second pipetting device being arrangedsymmetrically along one or two lines, and the tips constituting thethird pipetting device being arranged symmetrically along two lines. 30.Set of reaction vessels according to claim 28, wherein the firstpipetting device, the second pipetting device and the third pipettingdevice constitute at least one different pipetting device.
 31. Reactionvessel according to claim 23, wherein the angle is between about 60 and150°.
 32. Reaction vessel according to claim 23, wherein the angle isbetween about 80 and 120°.
 33. Reaction vessel according to claim 25,wherein the ratio is between about 1:5 to 1:20.
 34. Reaction vesselaccording to claim 25, wherein the ratio is about 1:10.